A Green Analytical Method for Simultaneously Determining Plasticizers Residues in Honeys from Different Botanical Origins: An Interview with Silvia Valverde and Ana María Ares

Recent research by the Analytical Chemistry Group of the University of Valladolid (Spain) has focused on determining nine plasticizers in honey samples by gas chromatography–mass spectrometry (GC–MS). While the team points out that there have been studies conducted previously focusing on the assessment of other contaminants in honey (such as metals, pesticides, antibiotics and veterinary drugs), there are comparatively few that have been done regarding plastics specifically. LCGC International spoke to Silvia Valverde and Ana María Ares of this group about their research and the resulting article.

Honeycomb with honey on white background. © Dionisvera - stock.adobe.com

You mention in your paper (1) that there have been few studies focused on assessing the presence of plastic contaminants in honey in the past. Why do you think this is? Are there any that were especially inspirational in your research?

Despite plastics having been used for decades, our awareness of their pollution and widespread presence is relatively recent, as is our understanding of their adverse health effects. The studies conducted by Peñalver and associates (2), Kartalovic and co-authors. (3) and Di Fiore and associates (4), have served as inspiration for this research. These studies have played a crucial role in advancing research on the presence of contaminants, particularly plastics, in diverse environmental and food contexts, such as honey.

While it goes without saying that we shouldn’t want plasticizers in our honey, do their presence or other environmental chemicals affect the bee, the hive, or the honey in any way?

The presence of plasticizers and other pollutants in honey can negatively impact bees and the hive by weakening their immune systems and interfering with their behavior and reproduction. This can lead to a decline in the beehive population. Additionally, these contaminants can compromise the quality and safety of honey for human consumption by altering its taste and potentially making it toxic. Therefore, it is essential to monitor and minimize honey contamination, as well as the factors exposed to bees.

Which plasticizers were the most prevalent and what is their source in the environment?

The most prevalent plasticizers in our study, BBP (benzyl butyl phthalate), DEP (diethyl phthalate), and DEHA (di-2-ethylhexyl adipate), can reach honey through various environmental pathways:

1. Soil and water contamination. Plasticizers can seep into soil and water near sources of contamination such as factories or landfills. Honeybees may collect contaminated water or nectar from plants growing in polluted soil.
2. Contaminated air: Plasticizers can disperse in the air, and honeybees can encounter these contaminants during their flight and bring them back to the beehive.
3. Deposition on plants: Plasticizers from air and water can settle on plants. Honeybees gather nectar and pollen from these plants, transferring contaminants into the honey.
4. Use of contaminated products: Products containing plasticizers, like plastics in hive structures or agricultural practices nearby, can directly transfer these chemicals to honey through contact.

Why were gas chromatography (GC) and high performance liquid chromatography (HPLC) your techniques of choice in your analysis?

GC is particularly suitable for analyzing phthalates due to its ability to separate volatile organic compounds based on their vapor pressures. Phthalates, being volatile, can be effectively detected by GC at very low concentrations, which is critical for assessing contaminants in food products like honey where even trace amounts matter. GC offers rapid analysis, facilitating high sample throughput, and ensures excellent quantitative accuracy, essential for precisely measuring phthalate levels in honey samples.

How are the analytical methods you selected and developed environmentally friendly or so-called green in their approach?

An attempt has been made to develop an analytical methodology that is as environmentally friendly as possible by using ethyl acetate as an extractant, known as a green solvent, and employing minimal volumes (< 5 mL) of this solvent. The sustainability of this approach has been evaluated using several green analytical metrics, with the most significant drawback identified as the energy consumption of the GC–MS instrumentation, which is essential for these analyses and unavoidable.

Briefly state your findings in this study.
The study developed a novel analytical method using gas chromatography-mass spectrometry (GC–MS) to analyze nine plasticizers in honey samples. Efficient sample treatment involving dual solvent extraction with ethyl acetate and cleanup with florisil resulted in satisfactory analyte recoveries (77% to 118%). Chromatographic analysis on an Agilent HP-5MS column demonstrated quick analysis times (< 21 minutes) under optimized temperature conditions. The method was validated for selectivity, low limits of detection (0.1–3.1 μg/kg), quantification (0.2–10.3 μg/kg), linearity, matrix effect, trueness, and precision (relative standard deviation < 9%). Analysis of thirty samples from various sources revealed residues of five plasticizers in most samples. Importantly, health risk assessment based on detected levels indicated no significant risks to consumers.

You analyzed honeys from three different botanical origins (multifloral, rosemary, and heather). Were there any types of honey more susceptible to plasticizers or other environmental contaminants? Were there any other variables, like hive location, that influenced their presence?

This study, based on the analysis of 30 honey samples, provides initial insights that necessitate a more comprehensive investigation for conclusive results. Initial indications suggest that multifloral honeys might have higher concentrations of contaminants, while honeys sourced from local markets show potentially higher levels compared to those from experimental fields. However, no consistent pattern based on hive location has been discerned. As emphasized, a larger and more extensive study is required to draw definitive conclusions regarding the susceptibility of different honey types to plasticizers contaminants.

Do your findings correlate with what you had hypothesized?

The findings generally align with our hypotheses. We anticipated detecting residues of plasticizers in honey samples due to potential environmental exposures. The efficient sample treatment method we developed allowed us to detect five specific plasticizers across various honey sources, confirming our expectations. However, the precise distribution and levels of these contaminants exceeded our initial predictions, highlighting the need for robust analytical methods to assess environmental and health impacts accurately.

Was there anything particularly unexpected that stands out from your perspective?

During the optimization of sample treatment, a strong matrix effect was observed across all three botanical origins evaluated, which could be effectively minimized with appropriate clean up steps. It was surprising to find that all origins exhibited a similar response to this matrix effect, a phenomenon not commonly observed with other contaminants studied. Regarding the results, it was unexpected to detect the presence of phthalates in all samples, specifically three of them, regardless of the type and origin of the honey.

Were there any limitations or challenges you encountered in your work?

In terms of challenges, analyzing phthalates presents a significant analytical hurdle due to their widespread presence in common laboratory materials. It requires meticulous cleaning of all equipment to ensure they are free from these contaminants, thus preventing false positives. Moreover, as previously mentioned, the limited number of samples analyzed underscores the need for a larger sample size to enhance the reliability of the conclusions drawn.

What best practices can you recommend in this type of analysis for both instrument parameters and data analysis?

Cleanliness in sample preparation, including rigorous cleaning of equipment and selecting efficient sample treatment methods, helps minimize contamination and ensure accurate measurements.

It is recommended to use statistical methods such as calculating mean concentrations, standard deviations, and performing ANOVA to effectively compare sample groups and identify significant differences. Additionally, employing visual tools like box plots is advised to illustrate data distribution and outliers, enhancing the interpretation of complex datasets such as those from contaminant assessments in various types of honey.

Are there any other natural food crops that you think might benefit from this sort of analysis?

This analytical approach could be beneficial for assessing a wide range of natural food crops. It could be particularly valuable for crops exposed to environmental contaminants or those requiring strict quality control measures. Examples include fruits, vegetables, grains, and herbs often subjected to pesticides and heavy metals.

Can you please summarize the feedback that you have received from others regarding this work?

The project has sparked significant interest across various sectors of society, both within the scientific community and among the public. This has led to its presentation at several international conferences and its coverage as a notable news story in newspapers in Spain.

Do you think more of this type of analysis should be performed on other natural foods?

Additionally, products like milk, meat, and seafood could also benefit from rigorous contaminant analysis to uphold food safety standards. Such comprehensive analyses are essential for ensuring consumer health and maintaining the integrity of food products worldwide.

What are the next steps in this research and are you planning to be involved in improving this technology?

The next steps would involve expanding the study to clarify conclusions regarding the accumulation of plasticizers in honeys, exploring differences based on botanical and geographical origins, as well as packaging of the product.

References

1. Fuente-Ballesteros, A.; Bernal, J.; Ares, A. M.; Valverde, S. Development and Validation of a Green Analytical Method for Simultaneously Determining Plasticizers Residues in Honeys from Different Botanical Origins. Food Chem. 2024, 455, 139888. DOI: 10.1016/j.foodchem.2024.139888

2. Peñalver, R.; Arroyo-Manzanares, N.; Campillo, N.; Viñas, P. Targeted and Untargeted Gas Chromatography-Mass spectrometry Analysis of Honey Samples for Determination of Migrants from Plastic Packages. Food Chem. 2021, 334, 127547. DOI: 10.1016/j.foodchem.2020.127547

3. Kartalovic, B.; Vranešević, J.; Petrović, J.; Đurđević, B.; Ratajac, R. Detection of Microplastic Residues-Developing a Method for Phthalates in Honey. Archives of Veterinary Medicine 2021, 14 (2), 19–33. DOI: 10.46784/eavm.v14i2.285

4. Di Fiore, C.; De Cristofaro, A.; Nuzzo, A.; Notardonato, I.; Ganassi, S.; Iafigliola, L.; Sardella, G.; Ciccone, M.; Nugnes, D.; Passarella, S.; Torino, V.; Petrarca, S.; Di Criscio, D.; Ievoli, R.; Avino, P. Biomonitoring of Polycyclic Aromatic Hydrocarbons, Heavy metals, and Plasticizers Residues: Role of Bees and Honey as Bioindicators of Environmental Contamination. Environ. Sci. Pollut. Res.2023, 30 (15), 44234–44250. DOI: 10.1007/s11356-023-25339-4

Ana María Ares (Associate Professor in Analytical Chemistry, researcher and secretary of TESEA group, University of Valladolid-UVa, left) has contributed for expanding the knowledge about some relevant issues such as food safety and quality, including the analysis of contaminants and bioactive compounds, and in development of new analytical strategies, especially those that involved green chemistry. She studied chemistry at UVa and received her PhD degree in 2015 at the same institution. After five years working as postdoctoral researcher in Analytical Development and Validation in a pharmaceutical industry and continued linked as professor at UVa, she obtained a full position in the same institution. In the last years, she has mainly worked in the apiculture area, especially in developing new strategies for determining pesticides and bioactive compounds in bee products. During her scientific career, she has worked with several analytical separation techniques as HPLC, GC, CE, and SFC coupled to several different detectors. She has also contributed as author/co-author to more than 60 scientific publications, 30 congress presentations, 5 book chapters, and 1 patent. In addition, she has also participated as Principal Researcher/Researcher in 4 research projects or contracts with various industries and government agencies. She is also member of the Editorial Board of several journals. Dr. Silvia Valverde (Assistant Professor in Analytical Chemistry, University of Valladolid, right) specializes in the development and enhancement of analytical methodologies for bioactive, pesticide and pollutant compounds determination in agri-food matrices. She has a strong focus on chromatographic techniques (LC and GC) coupled with several detectors, specifically mass spectrometry and tandem mass spectrometry. As well as on sample treatment and extraction techniques. She completed her Ph.D. in 2018 at the University of Valladolid. Subsequently, she worked in the private sector and undertook a postdoctoral stage at the Autonomous University of Madrid, where she was closely associated with the fertilizer sector, collaborating with prominent companies such as Fertinagro Biotech SL, Tradecorp, and Tessenderlo Kerley. In 2022, she joined the Department of Analytical Chemistry at the University of Valladolid as an Assistant Professor, where she currently works. She has authored 30 scientific publications, 4 book chapters, and received over 500 citations. In addition to participating in nine research projects, both publicly funded and financed by recognized companies in the wine and fertilizer sectors. She serves as an external reviewer for several journals and continues to contribute significantly to the field of analytical chemistry. Photo courtesy of authors.